Vaccines for mycoplasma bovis and methods of use

ABSTRACT

The invention of novel, effective vaccines against  Mycoplasma bovis  for use in cattle is described. These vaccines demonstrate no undesirable side effects and protect against  M. bovis  related disease, such as contagious mastitis, respiratory pneumonia, joint infections, keratoconjunctivitis and middle ear infections. The novel vaccines also lessen the effect of  M. bovis  infections on milk production, weight gain and animal health. Methods of diagnosing, characterizing and treating  M. bovis  infections as specific biotypes are also disclosed. Vaccine compositions made in accordance with the invention may be either of the attenuated or inactivated variety. Vaccines may also include antigens from other pathogens so as to provide a protective immunogenic response to diseases other than those caused by  M. bovis.

This application claims the benefit of priority of U.S. ProvisionalApplication No. 60/164,286, filed Nov. 8, 1999, the entire contents ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel vaccines for protection againstMycoplasma bovis disease in animals, compositions for the diagnosis ofsuch infections, and methods of diagnosis and vaccination.

BACKGROUND OF THE INVENTION

Mycoplasma bovis is a pathogenic prokaryote belonging to a class oforganisms that is intermediate in size between a bacteria and virus.These mycoplasmas are the smallest of the free-living microorganisms.They are characterized by the lack of a cell wall and are enveloped withonly a cell membrane, which allows for varying morphological shapes andunique growth requirements.

Mycoplasmas are known to cause infectious disease in most species ofanimals. In bovine species, Mycoplasma bovis is an opportunisticmicroorganism that causes infectious disease that is of significanteconomic importance to the livestock industry. Mycoplasma bovisisolation in a diseased bovine can be the result of its role as either aprimary or secondary causative etiological disease agent. Clinicaldisease and losses associated with infections caused by Mycoplasma bovisin beef and dairy cattle include: contagious mastitis, respiratorypneumonia, joint infections (arthritic conditions),keratoconjunctivitis, and middle ear infections. Even though severalspecies of mycoplasmas have been isolated in cattle, by far the mostprevalent is Mycoplasma bovis. For mastitis infections, the prevalenceof M. bovis is reported to be 70% or more.

Diseases caused by mycoplasmas are often resistant to antimicrobialtherapy, leaving no effective means of treatment. Consequently, the onlyeffective control method is to cull animals from a herd. This hasenormous economic implications in the dairy industry where losses aremeasured by the value of the culled animals as well as the impact onboth milk quality and quantity due to clinical and subclinicalinfections. Mycoplasma infections resulting in bovine mastitis areincreasing in prevalence and geographical distribution. In the UnitedStates, this higher prevalence is due to a larger and more intensecattle production industry in which herds are rapidly expanding, placingthem at greater risk. Increased incidence of M. bovis infection andrelated infectious disease in dairy herds has been noted worldwide(Jasper, D E 1982, J. Amer. Vet. Med. Assn. 181:158-162).

Control of disease caused by mycoplasmas in swine and avian species hasoccurred in recent years as the result of successful vaccinationprograms using safe and efficacious products. The design and developmentof an effective commercial vaccine in the United States to controlMycoplasma bovis has not yet occurred, even though changes in cattleproduction methods and husbandry practices have resulted in a greatercommercial need to control this agent from both an economic and foodquality perspective. Although there have been numerous attempts toproduce vaccine to protect against Mycoplasma bovis, the resultingexperimental vaccines have been deemed unacceptable due to the lack ofprotection as well as unacceptable site reactions in vaccinated animals(Boothby, et al. 1986 Cornell Vet 76: 188-197; Boothby et al. 1987 CanJ. Veterinary Research 51:121-125; Howard et al. 1987 Veterinary Record121:372-376; Boothby, et al. 1988 Can J. Veterinary Research52:355-359). Thus, there remains a need in the veterinary and animalhealth profession to provide a safe and effective vaccine to preventinfectious disease caused by Mycoplasma bovis with no unfavorable hostanimal reactions.

SUMMARY OF INVENTION

The invention disclosed herein provides safe and effective vaccines forthe prevention of Mycoplasma bovis disease in cattle. Also disclosed aremethods for characterizing biotypes of Mycoplasma bovis in cattle, bulkmilk tanks, and barns.

DESCRIPTION OF FIGURES

FIG. 1 is an illustration of the gel electrophoretic pattern for DNAproducts produced by Polymerase Chain Reactions from differentMycoplasma bovis isolates. On the left side of the figure, molecularweight standards based on restriction endonuclease digests of lambda andphi phage are shown. The size of the bands in the standard digests are,from top to bottom, for lambda; n/d, n/d, n/d, 2027, 1904, 1584, 1375,947, 831 and 564 base pairs, and for Phi X174; 1353, 1078, 872, 603,310, 284/271, 234, 194 and 118 base pairs. The relative location ofPCR-generated markers for different biotypes, designated A and B, areshown in lanes 5-12 to the right of the standards. Arrows in lanes 5 and7 indicate the presence of three and two characteristic bands forbiotypes A and B, respectively.

FIG. 2 is an illustration of the gel electrophoretic pattern for DNAproducts produced by Polymerase Chain Reactions from a second set ofMycoplasma bovis isolates. On the left side of the figure, molecularweight standards based on restriction endonuclease digests of lambda andphi phage are shown. The size of the bands in the standard digests are,from top to bottom, for lambda; n/d, n/d, n/d, 2027, 1904, 1584, 1375,947, 831 and 564 base pairs, and for Phi X174; 1353, 1078, 872, 603 and310 base pairs. The relative location of PCR-generated markers fordifferent biotypes, designated A and C, are shown in lanes 5-11 to theright of the standards. Arrows in lanes 5 and 8 indicate the presence ofthe three and two characteristic bands for biotypes A and C,respectively.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification and in the claims, “a,” “an” or“the” can mean one or more, depending upon the context in which it isused.

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides avaccine composition which is protective against Mycoplasma bovis diseasein a bovine species, comprising one or more inactivated or attenuatedMycoplasma bovis biotype(s) and a pharmaceutically acceptable excipient.The term “inactivated,” also referred to as “killed,” means that themicroorganisms are treated by any of several means known to the art sothat they no longer grow or reproduce, but that the microorganisms arestill capable of eliciting an immune response in the target animal.Examples of inactivating agents are: formalin, azide, freeze-thaw,sonication, heat treatment, sudden pressure drop, detergent (especiallynon-ionic detergents), lysozyme, phenol, proteolytic enzymes,propiolactone, Thimerosal (see U.S. Pat. No. 5,338,543 Fitzgerald, etal.), and binary ethyleneimine (see U.S. Pat. No. 5,565,205 Petersen, etal.). In a specific embodiment, the Mycoplasma bovis strains used in thevaccine are inactivated with beta-propiolactone (BPL).

Alternatively, the M. bovis biotypes used in the vaccine can beattenuated. The term “attenuated,” also referred to as “modified live,”is intended to refer to a living biotype of Mycoplasma bovis which hasbeen attenuated (modified) by any of a number of methods known in theart including, but not limited to, multiple serial passage, temperaturesensitive attenuation, mutation, or the like such that the resultantstrain is relatively non-pathogenic to a bovine species. The modifiedlive strain should be capable of limited replication in the vaccinatedanimal and of inducing a protective immune response which is protectiveagainst disease caused by virulent or wild-type Mycoplasma bovis.

The term “pharmaceutically acceptable” means a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an animal along with the immunogenic material (i.e.inactivated or attenuated M. bovis biotypes) without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the vaccine in which it iscontained. Examples of such pharmaceutically acceptable excipientsinclude water and physiological saline (for further examples, see Arnon,R. (Ed.) Synthetic Vaccines I:83-92, CRC Press, Inc., Boca Raton, Fla.,1987).

The invention disclosed herein is based in part on the discovery thatMycoplasma bovis infections in the field comprise mixtures of biotypes.The term “biotype” means a variant of a species, i.e. a strain, that canbe distinguished by one or more characteristics, such as ribosomal RNAsequence variation, DNA polymorphisms, serological typing, or toxinproduction (see e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989; DNA cloning: A Practical Approach, Volumes I and II, Glover,D. M. ed., IRL Press Limited, Oxford, 1985; Harlow and Lane, Antibodies,A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988)).

In another aspect of this invention, to produce an effective vaccineagainst Mycoplasma bovis, the vaccine must contain antigen derived froma biotype of Mycoplasma bovis. Examples of specific embodiments wouldinclude vaccines containing antigen derived from M. bovis biotypes A, B,or C. In a further specific embodiment, the vaccine comprises antigenderived from more than one M. bovis biotypes (e.g., A and B, A and C, Band C, or A, B and C). In a further specific embodiment, the vaccinecomprises antigen derived from one or more M. bovis biotypes and antigenderived from another pathogen. In a further specific embodiment, thevaccine comprises inactivated or attenuated M. bovis biotype A, B or C.In a further specific embodiment, the vaccine comprises at least twoinactivated or attenuated M. bovis biotypes (e.g., A and B, A and C, Band C, or A, B and C). In a further specific embodiment, the vaccinecomprises at least one inactivated or attentuated M. bovis biotype withantigen derived from another pathogen. In a preferred embodiment, thevaccine comprises inactivated or attenuated M. bovis biotype A, asdefined herein, and at least one other biotype of M. bovis.

It is anticipated that additional biotypes of M. bovis may emerge andmay be isolated with continued animal production. Additional biotypescan be added to the vaccine as needed. It is a matter of routinepractice to sample bulk milk tanks and blood from cows to isolateMycoplasma bovis cultures. These cultures can then be biotyped accordingto any of several typing techniques, as listed hereinabove. Vaccines canbe formulated based on the prevalence of M. bovis biotypes present inthe environment. Autogenous vaccines, i.e. those vaccines for use on thefarm where the microorganisms are isolated, can be custom-designed tocontain all biotypes found on the farm, but not any other biotype.Vaccines developed for use by a mass market, i.e. those vaccinesproduced for general use on many different farms containing pre-selectedbiotypes, can also be developed, marketed and used.

In another aspect, this invention provides a vaccine comprising asingle, inactivated or attenuated Mycoplasma bovis biotype, apharmaceutically acceptable excipient, and a suitable adjuvant. In aspecific embodiment, the vaccine contains inactivated or attenuated M.bovis biotypes A, B or C or any mixture thereof and may further containantigens from other pathogens.

In a preferred embodiment, the inactivated vaccines of this inventionare produced from biotypes freshly isolated from infected animals orfrom cryopreserved biotype cultures freshly prepared from infectedanimals. In a preferred embodiment, the attenuated vaccines of thisinvention are produced from cultures of biotypes which have been treatedso as to retain a limited ability to replicate within the vaccinatedanimal, but which does not retain the ability to infect other animalsand cause mycoplasma-related disease. The preparation and use ofattenuated vaccines is well-known to practitioners of ordinary skill inthe art.

The inactivated or attenuated M. bovis biotype(s) may be furtherprocessed to fractionate and/or standardize the antigenic mass. Forexample, specific biotypes might be isolated from samples and combinedto form specific combinations of biotypes in specific ratios. Similarly,components from a specific inactivated or attentuated M. bovis biotypemight be fractionated and a subset of those fractions combined withsimilarly fractionated components of another biotype to standardize theantigenic component of the vaccine preparation and to optimize itsefficacy. In one embodiment, the antigenic components derived from asingle biotype are enriched by removing non-immunogenic components fromthe cells of the biotype. In another embodiment, the vaccinepreparations are standardized to provide a required minimum cell contentper formulated dose. In a preferred embodiment, the vaccine comprisinginactivated M. bovis biotype(s) is formulated to deliver at least 10⁸ M.bovis cell equivalents of each biotype per dose. A complete vaccinationof a bovine species comprises the administration of recommended doses.In a preferred embodiment, two such doses will be administered. In afurther preferred embodiment, three such doses will be administered. Inanother preferred embodiment, the vaccine comprising attenuated M. bovisbiotype(s) is formulated to deliver at least 10⁵ M. bovis cells perbiotype. It is understood by those skilled in the art that the criticalvalue in describing a vaccination dose is the total amount of immunogenneeded to elicit a protective response by the host animal to infectiousdisease caused by virulent or wild-type M. bovis. The number and volumeof doses used can be varied and are determined by the practitioner basedon costs and the need to avoid deleterious side effects in the animalcaused by the administration. For example, the volume of oneadministration typically does not exceed 2-5 milliliters. The number ofdoses of inactivated vaccine needed in adult animals is typically oneinitial dose followed by 1-2 additional doses and annual revaccination.The number of doses of attenuated vaccine in adult animals is oneinitial dose followed by a booster. Subsequently, annual boosters areadministered.

The vaccines of the present invention may further comprise antigenicmaterial of other viruses and/or microorganisms known to be bovinepathogens, including, but not limited to, attenuated (modified-live) orinactivated viruses or microorganisms. Such combination vaccines provideprotection against a plurality of diseases to which the bovine speciesare exposed, including but not limited to immunogenic compositions forStaphylococcus aureus, Pasteurella hemolytica, Pasteurella multocida,Hemophilus somnus, Bovine Respiratory Syncytial Virus, Bovine DiarrheaVirus, E. coli and Infectious Bovine Rhinotrachial Disease.

In other embodiments, the vaccine of this invention further comprises asuitable adjuvant. As used herein, an “adjuvant” is a potentiator orenhancer of the immune response. The term “suitable” is meant to includeany substance which can be used in combination with the vaccineimmunogen (i.e. inactivated or attenuated M. bovis biotypes or fractionsthereof) to augment the immune response, without producing adversereactions in the vaccinated animal. Effective amounts of a specificadjuvant may be readily determined so as to optimize the potentiationeffect of the adjuvant on the immune response of an animal vaccinated.In a preferred embodiment, adjuvanting of the vaccines of this inventionis a 2-stage process utilizing firstly a 2% aluminum hydroxide solutionand secondly a mineral oil. In specific embodiments, suitable adjuvantscan be chosen from the following group: mineral, vegetable or fish oilwith water emulsions, incomplete Freund's adjuvant, E. coli J5, dextransulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain syntheticpolymers such as Carbopol (BF Goodrich Company, Cleveland, Ohio),poly-amino acids and co-polymers of amino acids, saponin, carrageenan,REGRESSIN (Vetrepharm, Athens, Ga.), AVRIDINE (N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), long chainpolydispersed β (1,4) linked mannan polymers interspersed withO-acetylated groups (e.g. ACEMANNAN), deproteinized highly purified cellwall extracts derived from non-pathogenic strain of Mycobacteriumspecies (e.g. EQUIMUNE, Vetrepharm Research Inc., Athens Ga.), Mannitemonooleate, paraffin oil, and muramyl dipeptide.

In another aspect, this invention discloses a method for immunizingbovine animals against infectious disease caused by Mycoplasma boviscomprising administering to a bovine animal immunogenic amounts ofinactivated or attentuated Mycoplasma bovis biotypes to elicit aprotective immune response by the animal. Preferably, the methodcomprises administering at least two inactivated or attenuatedMycoplasma bovis biotypes to elicit a protective immune response by theanimal. Immunization may be performed orally, intranasally,intratracheally, intramuscularly, intramammarily, subcutaneously,intravenously, or intradermally. The vaccine containing the inactivatedor attenuated M. bovis biotypes can be administered by injection, byinhalation, by ingestion, or by infusion. Repeated doses of the vaccinepreparations, i.e. “boosters”, are preferable at periodic time intervalsto enhance the immune response initially or after a long period of timesince the last dose. The time interval between vaccinations variesdepending on the age and condition of the animal. For lactating andadult animals, the first vaccination is preferably given at the end ofthe lactation cycle (i.e. “dry-off”), followed by a “booster” dose 2-4weeks later, and preferably followed by a second booster dose 2-4 weeksthereafter. Newborn calves are preferably vaccinated at birth, followedby booster doses every 3-5 weeks until the calves are 4-6 months old andannually thereafter. However, at risk or exposed stocker and feederanimals should be vaccinated more often, preferably no less than onceevery 6 months.

In another embodiment of the methods of this invention, the multiple M.bovis biotypes comprising the vaccine can be delivered in separateadministrations to the animal. For example, the vaccine comprisinginactivated M. bovis biotypes A and B can be delivered by separatelyadministering an immunogenic amount of biotype A in one injection and animmunogenic amount of biotype B in another injection. In a furtherembodiment, each separately administered biotype can be administered asa combination vaccination, comprising antigenic material of otherviruses and/or microorganisms known to be bovine pathogens.

The term “immunogenic amount” means an amount of an immunogen, i.e. theinactivated or attenuated M. bovis biotype(s) or a portion thereof,which is sufficient to induce an immune response in a vaccinated bovinespecies and which protects the animal against disease caused bywild-type or virulent M. bovis infections upon exposure thereto or whichhas a commercially beneficial effect that lessens the effect of M. bovison milk production, weight gain or animal health. In a preferredembodiment, bovine animals are immunized by administering at leastapproximately 10⁸ M. bovis cell equivalents of each inactivated biotypein the vaccine. In a specific embodiment, animals are immunized byadministering at least approximately 10⁸ M. bovis biotype A cellequivalents and approximately 10⁸ M. bovis biotype B cell equivalents,which have been inactivated, in at least two injections. In anotherspecific embodiment, bovine animals are immunized by administering atleast approximately 10⁸ M. bovis biotype A cell equivalents, 10⁸ M.bovis biotype B cell equivalents and approximately 10⁸ M. bovis biotypeC cell equivalents, which have been inactivated, in at least twoinjections.

In another preferred embodiment, bovine animals are immunized byadministering at least approximately 10⁵ M. bovis cells of eachattenuated biotype in the vaccine. In a specific embodiment, bovineanimals are immunized by administering at least approximately 10⁵ Mbovis biotype A attenuated cells and at least approximately 10⁵ M bovisbiotype B attenuated cells. In another specific embodiment, bovineanimals are immunized by administering at least approximately 10⁵ M.bovis biotype A cells, 10⁵ M. bovis biotype B cells, and 10⁵ M. bovisbiotype C cells.

In another aspect, this invention discloses a method for producing aMycoplasma bovis vaccine comprising contacting at least two liveMycoplasma bovis biotypes with an inactivating material andincorporating the inactivated Mycoplasma bovis biotypes into apharmaceutically acceptable excipient with a suitable produce aMycoplasma bovis vaccine. In a preferred method, selected Mycoplasmabovis biotypes are grown separately as pure cultures, free ofcontamination by viruses, bacteria or any other microbial agent,including other biotypes of M. bovis, to the desired cell equivalents,inactivated as described herein, and then combined in equal amounts witha pharmaceutically acceptable excipient to produce a Mycoplasma bovisvaccine. Alternatively, the biotypes can be grown together as a mixedculture to the desired cell equivalents, inactivated and then,optionally, combined with a pharmaceutically acceptable excipient and asuitable adjuvant to produce a Mycoplasma bovis vaccine.

In a further embodiment of the hereinabove disclosed method of producinga Mycoplasma bovis vaccine, the inactivated or attenuated Mycoplasmabovis biotypes are mixed with a suitable adjuvant. In a preferredmethod, the suitable adjuvant is an aluminum hydroxide-oil emulsion.

Selected M. bovis biotypes may be used as the basis for diagnostic toolsto detect the presence of M. bovis. In one aspect of this invention,samples from cattle would be tested for the presence of antibodiesspecific for M. bovis by contacting the samples with M. bovis cells orantigens derived from M. bovis. Examples of technologies that could beadapted to such a method include, but are not limited to, RIA, ELISA andimmunoblot. Examples of specific embodiments would include antigensderived from one or more M. bovis biotypes (e.g., A, B, C, A and B, Band C, A and C, or A, B and C). In a preferred embodiment, antigen fromeach of the M. bovis biotypes A, B and C would be utilized to test forthe presence of antibodies specific for each of the M. bovis biotypes,thus allowing an autogenous vaccine to be administered. In anotherembodiment, antibodies raised against M. bovis biotypes or antigensderived from selected biotypes would be used to test for the presence ofM. bovis biotypes A, B and C. Examples of specific embodiments wouldinclude antibodies reactive to antigens derived from one or more M.bovis biotypes (e.g., A, B, C, A and B, B and C, A and C, or A, B andC). In another embodiment, antigens derived from different biotypeswould be utilized to test for the presence of antibodies specificagainst antigens from a given biotype.

In a further embodiment, the present invention provides an isolatedMycoplasma bovis biotype A, Mycoplasma bovis biotype B, Mycoplasma bovisbiotype C, or any combination thereof.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES Example 1

Characterization and Typing of Field Isolates

Strains were collected from infected animals or milk tanks. Singlecolonies were cultured, and each culture was analyzed for cytotoxicityand for the presence of specific DNA markers by PCR fingerprinting.

PCR fingerprinting: Arbitrarily-chosen primers were selected; Primer 1and Primer 2 below (N representing deoxyinosine and A, C, T and Grepresenting the four naturally-occurring bases of DNA): [SEQ ID NO: 1]Primer 1: 5′ NNN NCG NCG NCA TCN GGC 3′; and [SEQ ID NO: 2] Primer 2:5′ NCG NCT TAT CNG GCC TAC 3′;

Mycoplasma bovis DNA was isolated and amplified, using these primers, ina polymerase chain reaction (PCR) as follows: The initial cycling stepwas for 120 seconds at 94° C. Denaturation was for 30 seconds at 94° C.,followed by annealing for 90 seconds at 40° C., extension for 120seconds at 72° C., with a final extension for 240 seconds at 72° C. Atotal of 35 cycles of amplification were used.

The resulting DNA products of the PCR were separated by non-denaturing1.5% agarose gel electrophoresis and were visualized by staining withethidium bromide and illuminating the gel with a UV light source.Comparison of the resultant patterns, characteristic for a givenbiotype, with molecular weight standards such as the EcoRI/HindIIIdigest of lambda phage or the HaeIII digest of phiX174 phage,electrophoresed alongside the PCR products, allows consistent andreproducible biotyping of M. bovis strains. Examples of biotypingresults using this method are provided below. Strain ID Culture # %Cytotoxicity Biotype Profile BA2580 1 95 A 2 0 A BA2491 1 82 A 2 100 A498 1 100 A 3 91 A 4082 1 100 B 2 91 B 3 83 B Tank 2-18 1 90 A 2 100 A 3100 A Tank 2-19 1 100 A 2 20 A 3 100 A L-56291 1 100 A 2 100 A 3 86 AL-477 1 84 C 2 76 C 3 90 C L-53219 1 66 A 2 100 A 3 100 A

Both cytotoxic (i.e. ≧40% cytotoxic) and noncytotoxic strains arepathogenic. While the majority of isolates are homogeneously cytotoxic,a few isolates, e.g. BA2580 and Tank 2-19, are a mixture ofnon-cytotoxic and cytotoxic colonies. Following extensive passage inculture, all strains become noncytotoxic, while passage through calvesaccentuates the initial phenotype, whether non-cytotoxic or cytotoxic.The PCR fingerprints for three M. bovis biotypes are illustrated inFIGS. 1 and 2 alongside the standards formed by the EcoRI/HindIIIrestriction endonuclease digest of lambda phage and the HaeIIIrestriction endonuclease digest of phi phage. The sizes of the resultantstandard fragments, in base pairs, are listed in the description ofFIGS. 1 and 2. A blank and a positive control for the PCR fingerprintingreactions are included in lanes 3 and 4, respectively, for both FIGS. 1and 2. Further description of PCR fingerprinting can be found inArtiushin et al. Int. J. Syst. Bacteriol. 46: 324-328 (1996), Fan et al.Avian Dis. 39: 729-735 (1995) and elsewhere. Other methods of biotypingmycoplasma, or other microorganisms, are well-known to the art and mayalso be used in the practice of the invention ( see e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989; DNA cloning: A PracticalApproach, Volumes I and II, Glover, D. M. ed., IRL Press Limited,Oxford, 1985; Harlow and Lane, Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, N.Y. (1988)).

Example 2

Preparation of an Inactivated Vaccine against Biotypes of Mycoplasma

A Mycoplasma bovis vaccine is prepared by inactivating a selectedbiotype of Mycoplasma bovis and combining this preparation with anadjuvant.

A. Selection of M. bovis biotypes

Isolates of M. bovis were obtained from samples of infected milk. Theseisolates were then cultured using standard techniques, such as thosedescribed by Knudtson et al. Vet. Microbiol. 11: 79-91 (1986).

Selected isolates were further expanded and characterized by biotype.Cultures of isolates representative of the characteristic biotypes, asdetermined by PCR fingerprinting, were selected and stocks of thesebiotypes were preserved by combining them with a gelatin proteinhydrolysate stabilizing solution and subjecting the product tocryopreservation. Pure biotype cultures were used to inoculate acontrolled fermentation of the microorganism for use in producingvaccine. PCR fingerprinting of these cultures for vaccine productionconfirmed their purity as single biotypes. Subsequent testing of thecryo-preserved stock(s) was performed in a USDA licensed facilityaccording to Title 9 Code of Federal Regulations to validate purity andidentity of the culture(s). Identity was determined to be Mycoplasmabovis by two independent laboratories when tested by indirectimmunofluorescence with specific antisera to the following species:

-   -   Mycoplasma bovis    -   Mycoplasma califoricum    -   Mycoplasina alkalescens    -   Mycoplasma canadense    -   Mycoplasma bovigenitalium    -   Mycoplasma bovirhinis    -   Mycoplasma arginnii    -   Acholeplasma laidlawii        B. Propagation of the Pure, Isolated Biotypes

Selected strains, or biotypes, identified as being pure, were propagatedin a defined media and further processed to make vaccines.

Mycoplasma bovis biotypes can be propagated in a variety of differentgeneral purpose, growth-promoting, defined media that are known to thoseknowledgable in the art, including, but not limited to, Hayflick Media,Adler Media, and Gourlay Media. In a preferred embodiment of thisinvention, the propagation medium is:

-   -   Yeast Extract: 5 grams per liter    -   Proteose Peptone: 2 grams per liter    -   Mixed substrate        -   Peptone, such as        -   Enhancetone: 20 grams per liter    -   Dextrose: 2 grams per liter    -   Sodium Chloride: 5 grams per liter    -   Sodium Phosphate: 2.5 grams per liter    -   Glycerol: 1 gram per liter    -   Nutrient        -   Horse Serum: 50 ml per liter    -   1% NAD/Cysteine: 20 ml per liter    -   Water: to volume of 1000 ml

Cultures were expanded and inoculated into media at a concentration of10⁷-10⁸ cfu/milliliter. Cultures were grown at a temperature between 30and 41° C., under normal atmospheric oxygen pressure, with thepercentage of CO₂ in the environment kept between 0% and 10%. Incubationtimes ranged from 8 hours to 72 hours. The endpoint of incubation isdetermined by the time at which the cultures reach stationary phase, asmeasured by standard microbiological methods.

Standard microbiological methods are used to determine immunogen mass,e.g. a direct plate count procedure or a spectophotometric opticaldensity method based upon light absorbance of the Mycoplasma boviscultured cell mass.

C. Inactivation of Mycoplasma bovis

Beta-propiolactone (BPL) is prepared as a 10% solution (v/v) in chilledwater. The chilled solution is slowly added to the M. bovis culture(s)with constant stirring, thereby allowing hydrolysis. This BPL solutionis added in the amount of 10 milliliters per liter of M. bovisculture(s). The pH of the BPL-M. bovis suspension is maintained between6.5 and 7.8, by adding sodium hydroxide as needed. The suspension iswarmed to room temperature and continuously agitated for 24 hours. Thecells are concentrated by centrifugation at 8,000 g or byultrafiltration.

D. Adjuvanting and Formulation of Vaccine

Adjuvanting and final formulation of bulk concentrated inactivated M.bovis cultures were done concurrently as described in followingprotocol:

-   -   1) Determine the final volume batch quantity desired based upon        2 milliliters per dose. Quantity of each ingredient to be added        is then calculated as described in steps 2 through 5.    -   2) Dispense an amount of inactivated M. bovis cell concentrate        necessary to contain a protective dose quantity sufficient for        the total number of doses being formulated, based on the cell        counts determined in the live culture.    -   3) Dilute the inactivated M. bovis cell concentrate with 0.85%        saline solution sufficient to bring the batch to the final        desired volume (following addition of adjuvant components)    -   4) Adjust pH to 6.0 to 6.5 using a 10 normal hydrochloric acid        solution.    -   5) Add an amount of 2% aluminum hydroxide solution to yield a        final formulated concentration of 8% to 16%; incubate for 24        hours.    -   6) Using 10 N sodium hydroxide solution, readjust the pH to 7.2        to 7.4    -   7) Emulsify mineral oil adjuvant with the diluted aluminum        hydroxide-absorbed inactivated M. bovis cells with an amount        sufficient to yield 4% to 12% in the final formulation.

Example 3

Preparation of Vaccine against M. bovis Biotypes A, B and C.

A Mycoplasma bovis vaccine was prepared containing antigen from 3biotypes; A, B and C. The process for preparation of vaccine from linewas the same as described for Example 2 above. Immunogenic componentsfrom biotypes A, B and C were combined after inactivation of selectedquantities of pure cultures of each biotype.

Example 4

Preparation of Vaccine against M. bovis Biotypes A, B, C and M.alkalescens.

Five lung and ear isolations were obtained from necropsied calves. Usingindirect immunofluorescence, the isolates were identified by Biomune as:

-   -   S99—0052—M. bovis—Lung    -   S99—0052—M. bovis—Ear    -   S99—0053—M. bovis—Lung    -   S99—0053—M. alkalescens—Lung.

Cultures were passaged 4× in Hayflicks modified liquid media withcharacterization and preparation of pure cultures.

Samples of cultures from isolates S99-0052 and S99-0053 were furthercharacterized and were determined to be pure M. bovis and M. alkalescensby an independent laboratory. Identity of isolate S99-0053 as M.alkascens was confirmed by further testing.

Two groups of isolates were further characterized. Cytotoxicity cellculture bioassays and PCR fingerprinting were performed. These assaysconfirmed the identification of the cultures, to be used for vaccineproduction, to be pure M. bovis and M. alkalescens.

From the isolated Mycoplasma, a vaccine containing antigens from M.bovis biotypes A, B, C and antigens from M. alkalescens was preparedusing the protocol described earlier.

Example 5

Field Trial of Vaccine against M. bovis Biotype A

Efficacy of an inactivated vaccine of this invention specific for M.bovis biotype A was determined under field conditions at a site with anendemic mycoplasma mastitis infection in the herd. An active fieldchallenge was confirmed, based on a historical review of cull cow lossesdue to M. bovis, farm site M. bovis environmental isolation records,cultural isolation of M. bovis from clinical mastitis cases in thenon-vaccinated animals, and isolation of M. bovis from dairy bulk tanks.Laboratory tests confirmed the identity of these isolations.

The dosage and regime protocol for field vaccinations were as follows:

-   -   Administration: 2 milliliter dose of an oil emulsion        adjuvanted M. bovis vaccine; subcutaneous injection in neck        region    -   Regime: 3 doses of vaccine    -   For lactating cows:        -   1st Vaccination at Dry Off (End of Lactation Cycle)        -   2nd Vaccination 2 to 3 Weeks Following 1^(st) injection        -   3rd Vaccination 2 to 3 Weeks Following 2^(nd) injection    -   For heifers:        -   The 3 doses are spaced 2-4 weeks apart prior to calving.            Preferably, the last dose is administered at least 10 days            prior to calving and the start of the lactation cycle.

Comparative results were used to measure efficacy of the vaccine.Samples taken from all animals presenting with clinical mastitis werecultured by an independent laboratory to monitor the absence or presenceof Mycoplasma bovis infection of the mammary gland. Field evaluationswere made by comparing clinical incidence of mastitis caused byMycoplasma bovis following herd vaccination to the base line herdincidence prior to vaccination. Results were as follows:

-   Pre Vaccination Base Line Incidence:

155 confirmed positive clinical Mycoplasma bovis infections

-   Post Vaccination Herd Incidence:    -   1^(st) year following vaccination:        -   24 confirmed positive clinical Mycoplasma bovis infections    -   2^(nd) year following vaccination:        -   1 confirmed positive clinical Mycoplasma bovis infection

No injection reactions were observed. No inflammatory udder reactionswere observed.

Animals were also evaluated for serological response using serumcollected from individual animals prior to and following the 2^(nd)vaccination. A direct ELISA was performed, with the following resultsfor selected animals: O.D. values Animal ID: Pre-vaccinationPost-vaccination 82651 .093 .313 82759 .189 .693 61043 .135 .273 3219.198 .586 83550 .495 1.733 9296 .289 1.553

An immune response is indicated when the post-vaccination values exceedthe pre-vaccination values by at least 2 fold.

Example 6

Field Trial of Vaccine against M. bovis Biotypes A, B and C

In the 3^(rd) calendar year of the trial described in Example 5,200replacement cows were introduced into the herd; 100 at the same site(Site 1)as for Example 5 and 100 into a second related site located inthe same geographical area (Site 2). Neither subset of replacement cowswere quarantined prior to being introduced to their respective groups.Within 2 months, serious problems with Mycoplasma mastitis were reportedat both Sites 1 and 2 by personnel at each site.

Testing of all cows at both sites, approximately 4,000 animalsaltogether, revealed the presence of 22 animals infected with M. bovis.Initial screening of all animals was accomplished by culturing pooledmilk samples (16 cows/sample). When pooled samples were identified aspositive for M. bovis by culturing milk, all animals in positive groupswere tested individually. Three independent studies confirmed isolationof “bovis species” and identification of the three different biotypes(A, B and C) of M. bovis was made by PCR fingerprinting. The PCRfingerprinting was carried-out as described above in Example 1.

A vaccine was prepared using antigen from 3 biotypes of M. bovis (A, Band C) as described in Example 3 above and was used to vaccinate cattleat both Site 1 and Site 2 according to the regime described in Example5. Vaccinations began in mid-September, 1999. The incidence ofMycoplasma mastitis was monitored by independent laboratory testing forthe presence of Mycloplasma in any animal determined by farm personnelto have mastitis.

Following vaccination of a significant portion of the herd at Site 1 andSite 2, the incidence of mycoplasma was greatly reduced. From Jan. 1,2000 to Jul. 18, 2000, there were only 10 animals reported positive forMycoplasma bovis at each site. This reduction in the incidence ofMycoplasma positive mastitis cows was regarded as a significantreduction by the operators of Sites 1 and 2. A breakdown of theincidence during Calendar year 2000 is as follows: Site 1. Site 2January 1 2 February 1 1 March — 3 April 3 1 May 1 2 June 1 — July 3 —

Example 7

Field Trial of Vaccine against M. bovis Biotypes A, B, C and M.alkalescens.

A vaccine prepared according to Example 4 comprising antigen from M.bovis biotypes A, B, C and M. alkalescens was used to vaccinate calvesat a large 17,000 head-calf raising facility. It has been determined bythe site's operators that Mycoplasma is a major respiratory problem.Sample bleedings and serological evaluation prior to initiation of thetrial indicated that approximately 50% of calves received areserologically negative as determined by direct ELISA bioassay.

Calves selected for the trial were bled and identified with numberedtags on Oct. 19, 1999. Serum was immediately collected. Each of thecalves was given the normal treatment regime for newborn cattle arrivingat the site (colostrum, etc.). In addition, the calves were vaccinatedthree times with 2 mL of the vaccine prepared in Example 4. Vaccine wasadministered approximately every 7 days for the first 3 weeks. On Nov.22, 1999, serum samples were taken from the 36 calves remaining of theoriginal 50. On Dec. 21, 1999, serum samples were taken from 35 of the36 calves remaining of the original 50 (one calf could not be located).

Response to vaccination was monitored using the ELISA bioassay usedearlier to determine the serological status of calves at the site priorto initiating the trial. A representative random sample of calves (16)which had been serologically negative at day zero was monitored at dayzero, post 2^(nd) vaccination and post 3^(rd) vaccination. A 7-foldincrease in the immunological response over their pre-vaccinated statuswas realized in the animals following the vaccination procedure. This isbelieved to be a significant response and the vaccine's efficacy wasconfirmed by the fact that the vaccinated animals performed well asmeasured by days to market and rate of gain, both important indicatorsof a calf's health and well-being.

Example 8

Field Trial of Vaccine against M. bovis Biotype B.

Biotype B was isolated from a 1200 cow Jersey dairy herd experiencingMycoplasma mastitis infections in the herd. A vaccine against Biotype Bwas prepared as described in Example 2 and used in a manner consistentwith the vaccination regime previously described. Following theinitiation of the vaccination regime for the herd in February, 2000, aveterinarian monitored the herd for the incidence of M. bovis. The dairyreported in September 2000 that there were no confirmed cases ofMycoplasma in vaccinated animals, despite the continued challenge fromthe presence of confirmed, infected nonvaccinated animals. As ofSeptember, approximately 50% of the herd had been vaccinated. Nounfavorable reactions resulting from the vaccine's use have beenreported.

Example 9

Field Trial of Vaccine against M. bovis Biotype C.

Biotype C was isolated from a cultured isolate derived from a Holsteindairy herd of approximately 400 animals. This herd had been experiencingMycoplasma mastitis infections and had been experiencing positive bulkmilk tanks for the year prior to May, 1999. In March, 1999, thirteencows had been identified as positive for Mycoplasma bovis infection. Avaccine specific for biotype C was made as described in Example 2 andwas used in a manner consistent with the vaccination regime previouslydescribed. The owner and herd health veterinarian monitored performanceof the vaccine. There have been no reported clinical mastitis events invaccinated animals. No unfavorable reactions in animals receiving theproduct have been reported.

1. A vaccine which is protective against Mycoplasma bovis clinicaldisease in a bovine species comprising at least one inactivated orattenuated Mycoplasma bovis biotype, an adjuvant, and a pharmaceuticallyacceptable excipient, wherein: the adjuvant does not include saponin,the clinical disease includes respiratory pneumonia, and the vaccinedoes not cause unfavorable reactions.
 2. The vaccine of claim 1, whereinthe Mycoplasma bovis biotype is inactivated.
 3. The vaccine of claim 1,wherein the Mycoplasma bovis biotype is attenuated.
 4. The vaccine ofclaim 1, wherein at least one of the inactivated or attenuatedMycoplasma bovis biotypes is selected from the group consisting ofbiotype A, biotype B, and biotype C.
 5. A vaccine which is protectiveagainst Mycoplasma bovis clinical disease in a bovine species comprisingat least two inactivated or attenuated Mycoplasma bovis biotypes and apharmaceutically acceptable excipient, wherein the vaccine does notcause unfavorable reactions.
 6. The vaccine of claim 5, furthercomprising a suitable adjuvant.
 7. The vaccine of claim 6, wherein theMycoplasma bovis biotypes are inactivated.
 8. The vaccine of claim 7,wherein the inactivated Mycoplasma bovis biotypes are geneticallydifferent as determined by an analysis of DNA or RNA from the biotypes.9. The vaccine of claim 10, wherein the analysis is by PCRfingerprinting, analysis of ribosomal RNA, or analysis of DNApolymorphisms.
 10. The vaccine of claim 9, wherein the analysis is byPCR fingerprinting.
 11. The vaccine of claim 10, wherein the PCRfingerprinting uses arbitrarily chosen primers.
 12. The vaccine of claim11, wherein the PCR fingerprinting uses as primers 5′ NNN NCG NCG NCATCN GGC 3′ (SEQ ID NO:1) and 5′ NCG NCT TAT CNG GCC TAC 3′ (SEQ IDNO:2).
 13. The vaccine of claim 8, wherein the at least two Mycoplasmabovis biotypes have been identified as being genetically differentbiotypes by a process comprising: (a) isolating DNA from the biotypes;(b) amplifying the DNA by PCR; (c) separating the amplified DNA by gelelectrophoresis; and (d) comparing the resulting patterns from the gelelectrophoresis to identify the different biotypes.
 14. The vaccine ofclaim 5, wherein the Mycoplasma bovis biotypes are attenuated.
 15. Thevaccine of claim 5, wherein the Mycoplasma bovis biotypes are selectedfrom the group consisting of biotype A, biotype B, and biotype C.
 16. Avaccine which is protective against Mycoplasma bovis mastitis in abovine species comprising at least one inactivated or attenuatedMycoplasma bovis biotype and a pharmaceutically acceptable excipient,wherein the vaccine does not cause unfavorable reactions.
 17. Thevaccine of claim 16, where the vaccine is protective against Mycoplasmabovis mastitis in a bovine species following systemic administration.18. The vaccine of claim 16, comprising at least two inactivatedMycoplasma bovis biotypes.
 19. The vaccine of claim 18, wherein thevaccine includes at least one inactivated Mycoplasma bovis biotypeselected from the group consisting of biotype A, biotype B, and biotypeC.
 20. The vaccine of claim 18, wherein the inactivated Mycoplasma bovisbiotypes are genetically different as determined by an analysis of DNAor RNA from the biotypes.
 21. The vaccine of claim 20, wherein theanalysis is by PCR fingerprinting, analysis of ribosomal RNA, oranalysis of DNA polymorphisms.
 22. The vaccine of claim 21, wherein theanalysis is by PCR fingerprinting.
 23. The vaccine of claim 22, whereinthe PCR fingerprinting uses arbitrarily chosen primers.
 24. The vaccineof claim 23, wherein the PCR fingerprinting uses as primers 5′ NNN NCGNCG NCA TCN GGC 3′ (SEQ ID NO:1) and 5′ NCG NCT TAT CNG GCC TAC 3′ (SEQID NO:2).
 25. The vaccine of claim 20, wherein the Mycoplasma bovisbiotypes have been identified as being genetically different biotypes bya process comprising: (a) isolating DNA from the biotypes; (b)amplifying the DNA by PCR; (c) separating the amplified DNA by gelelectrophoresis; and (d) comparing the resulting patterns from the gelelectrophoresis to identify the different biotypes.
 26. The vaccine ofclaim 16, wherein, when the vaccine is administered to a plurality ofcows in a herd of cows, the incidence of mastitis caused by Mycoplasmabovis in the herd before administering is greater than the incidence ofmastitis caused by Mycoplasma bovis in the herd after administering. 27.The vaccine of claim 26, wherein the vaccine is administered to at leastabout 50% of the herd.
 28. The vaccine of claim 27, where the vaccine isadministered together with an adjuvant.
 29. The vaccine of claim 1,wherein the adjuvant is an aluminum hydroxide-oil emulsion; a mineral,vegetable, or fish oil-water emulsion; a water-oil-water emulsion;incomplete Freund's adjuvant; E. coli J5; dextran sulfate; iron oxide;sodium alginate; Bacto-Adjuvant; a synthetic polymer; Carbopol; apoly-amino acid; a co-polymer of amino acids; saponin; carrageenan;REGRESSIN®; N,N-dioctadecyl-N′-N′-bis(2-hydroxyethyl) propanediamine; along chain polydispersed β(1,4) linked mannan polymer interspersed withO-acetylated groups; deproteinized cell wall extracts from anon-pathogenic strain of Mycobacterium; mannite monooleate; paraffinoil; or muramyl dipeptide.
 30. The vaccine of claim 2, wherein theMycoplasma bovis biotype has been inactivated by treatment with:formalin, azide, freeze-thawing, sonication, heat, sudden pressure drop,detergent, lysozyme, phenol, proteolytic enzymes, β-propiolactone,Thimerosal, or binary ethyleneimine.
 31. A vaccine which is protectiveagainst Mycoplasma bovis clinical disease in a bovine species comprisingat least one Mycoplasma bovis biotype that has been inactivated bytreatment with β-propiolactone.
 32. The vaccine of claim 31, wherein theclinical disease is Mycoplasma bovis mastitis.
 33. The vaccine of claim32, comprising at least two Mycoplasma bovis biotypes that have beeninactivated by treatment with β-propiolactone.
 34. The vaccine of claim33, wherein the at least two inactivated Mycoplasma bovis biotypes aregenetically different as determined by an analysis of DNA or RNA fromthe biotypes.
 35. The vaccine of claim 34, wherein the analysis is byPCR fingerprinting, analysis of ribosomal RNA, or analysis of DNApolymorphisms.
 36. The vaccine of claim 35, wherein the analysis is byPCR fingerprinting.
 37. The vaccine of claim 36, wherein the PCRfingerprinting uses arbitrarily chosen primers.
 38. The vaccine of claim37, wherein the PCR fingerprinting uses as primers 5′ NNN NCG NCG NCATCN GGC 3′ (SEQ ID NO:1) and 5′ NCG NCT TAT CNG GCC TAC 3′ (SEQ IDNO:2).
 39. The vaccine of claim 34, wherein the at least two Mycoplasmabovis biotypes have been identified as being genetically differentbiotypes by a process comprising: (a) isolating DNA from the biotypes;(b) amplifying the DNA by PCR; (c) separating the amplified DNA by gelelectrophoresis; and (d) comparing the resulting patterns from the gelelectrophoresis to identify the different biotypes.